The inhibitor-of-apoptosis protein (IAPs) family is characterized by one or more repeats of a highly conserved ˜70 amino acid domain termed the baculoviral IAP repeat (BIR) and suppress apoptosis triggered by a wide variety of stimuli, including viral infection, chemotherapeutic drugs, staurosporin, growth factor withdrawal, and by components of the TNF-a/Fas apoptotic signaling pathways (Deveraux, Q. L. and Reed, J. C. (1999) Genes & Dev. 13:239–252; LaCasse et al., (1998) Oncogene 17:3247–3259; Miller, L. K. (1999) Trends Cell Biol. 9:323–328). While first identified in baculovirus, the AP family has been conserved evolutionarily from viruses to nematodes, flies, and several mammalian species. There are currently five human IAP family members, c-IAP1, c-IAP2, XIAP, NAIP, and survivin (Ambrosini, et al., (1997) Nat. Med. 3:917–921; Duckett et al., (1996) EMBO J. 15:2685–2689; Liston et al., (1996) Nature 379:349–353; Rothe et al., (1995) Cell. 83:1243–1252). Recombinant expression of IAP proteins blocks apoptosis induced by various stimuli (Duckett et al. ibid; Liston et al. ibid), and promotes abnormally prolonged cell survival in the developmentally-regulated model of the Drosophila eye, in vivo (Hay et al., (1995) Cell. 83:1253–1262). All of the human IAP family members, with the exception of NAIP, have been shown to interact with specific cysteine proteases, or caspases, required for the cleavage of certain proteins involved in the disassembly of the cell during apoptosis (Thomberry and Lazebnik (1998) Science 281:1312–1316). The caspases are synthesized as inactive zymogen forms which upon apoptotic stimulation are proteolytically processed in sequential manner into their active heterotetrameric forms. c-IAP1, c-IAP2, XIAP, and survivin bind potently to the active forms of the terminal caspases-3 and -7, but do not interact with caspases-8, which is the most proximal caspase from the TNF-a/Fas receptor (Deveraux et al., (1997) Nature 388:300–304; Roy et al., (1997) EMBO J. 16:6914–6925; Tamm et al., (1998) Cancer Res. 58:5315–5320). In addition, c-IAP1, c-IAP2, and XIAP bind to the zymogen form of caspase-9 thereby preventing its proteolytic processing as well as the processing of proteases, such as caspase-3, -6, and -7 (Deveraux et al., (1998) EMBO J. 17:2215–2223). Abrogation of caspase activity, a common downstream component of apoptosis, enables IAPs to have widespread anti-apoptotic potential.
The BIR domain forms a novel zinc-fold that is the critical motif for their anti-apoptotic activity and interaction with caspases (Hinds et al. (1999) Nat. Struct. Biol. 6: 648–651). While many IAPs contain up to three tandem BIR repeats, a single BIR domain is sufficient for caspase interaction and protection from apoptosis (Tamm et al., ibid; Takahashi et al., (1998) J. Biol. Chem. 273:7787–7790). Many of the IAP proteins (c-IAP1, c-IAP2, XIAP, as well as viral and insect IAPs) also contain a RING domain near their COOH-termini. The role for the RING domain varies depending on the AP and/or the apoptotic stimulus, but does not appear to be required for the anti-apoptotic activity of human IAPs (Roy et al., ibid; Takahashi et al. ibid; Hay et al., (1995) Cell 83:1253–1262; Vucic et al., (1998) Mol. Cell. Biol. 18:3300–3309). Deletion of the RING domain in c-IAP2 has suggested a critical role in TNF-a-mediated NF-kB activation, thereby providing an additional mechanism for the IAPs anti-apoptotic activity (Chu et al., (1997) Proc. Natl. Acad. Sci. 94:10057–10062). However, it is unclear if this is a general feature of the RING domain in other IAP family members.
Several of the IAP family members have been reported to play a role in pathological conditions, particularly neurodegenerative disorders and cancer. For instance, the NAIP gene was originally identified based on its deletion in patients with spinal muscular atrophy (SMA), a neurodegenerative disorder characterized by motor neuron depletion through apoptosis (Roy et al., (1995) Cell 80:167–178). The correlation between NAIP, SMA, and apoptosis suggests that NAIP may be required for the survival of these neurons and that mutations in the NAIP locus contribute to SMA. In addition, NAIP levels are transiently elevated following ischemia and damage can be inhibited by overexpression of NAIP in vivo (Xu et al., (1997) Nature Med. 3:997–1004). Other IAPs, particularly survivin, have been correlated with cancer. Survivin is overexpressed in nearly all human tumors and transformed cell lines, but is rarely present in normal adult tissues (Ambrosini et al. ibid; Tamm et al., ibid; Grossman et al., (1999) Lab. Invest. 79:1121–1126; Kawasaki et al., (1998) Cancer Res. 58:5071–5074; Lu et al., (1998) Cancer Res. 58:1808–1812; Saitoh et al., (1999) Int. J. Oncol. 15:137–141), and XIAP, c-IAP1, and c-IAP2 are expressed in malignant gliomas (Wagenknecht et al., (1998) J. Biol. Chem. 273:11177–11182). Depletion of survivin using antisense or dominant negative mutants induce apoptosis implying that survivin expression contributes to the survival of cancer cells (Grossman et al. ibid; Ambrosini et al., (1998) J. Biol. Chem. 273:11177–11182; Li, F et al., (1999) Nature Cell Biol. 1:461–466; Li, F et al.,(1998) Nature 396:580–584).
Abberantly increased apoptosis or abnormally prolonged cell survival may both contribute to the pathogenesis of human diseases, including autoimmune disorders, neurodegenerative processes, and cancer (Steller (1995) Science 267:1445–1449).
Therapeutic and diagnostic uses of nucleic acids that encode various inhibitors of apoptosis relating to a member of the IAP family have been described in the patent literature. See for example, International Patent Publication Nos. WO 97/06255, WO 97/26331, WO 97/32601 and WO 98/22589. WO 98/22589 (Yale University) describes and claims the survivin gene/protein.
The present invention derives from the identification of a further IAP gene and protein which the inventors have termed livin. The full-length cDNA isolated and sequenced (SEQ ID NO. 1) of the human livin gene is 1376 nucleotides in length. The coding sequence is 843 nucleotides in length (SEQ ID No. 2) and the corresponding protein sequence is 280 amino acids in length (SEQ ID NO. 3). The BIR domain is located from about amino acids 87–154 and the RING domain from about amino acids 249–258. The overall protein similarity of livin to other IAP family members based on the GAP pairwise sequence alignment (GCG®, Madison, Wis.) was 31.7% to c-IAP1, 31.1% to c-IAP2, 46.4% to XIAP, 30.3% to NAIP, and 32.8% to survivin. At a structural level it was similar to survivin with respect to having just a single BIR domain. However, livin does not have a coiled-coil domain like survivin, but rather contains a COOH-terminal RING domain found in c-IAP1, c-IAP2, and XIAP. Expression of livin inhibited apoptosis by a number of stimuli, whereas an antisense construct was shown to induce apoptosis. Like its other family members, livin was capable of binding to caspases that correlated with its anti-apoptotic activity. Restricted expression of livin mRNA during development and transformed cell lines suggests a very specific role for livin.
Since livin, described infra, is present in certain cancer cell lines and prevents apoptosis it appears to be a significantly valuable therapeutic target for cancers exhibiting reduced apoptosis, such as melanomas as well as tissues effected by elevated apoptosis (for example, neurodegenerative diseases like Alzheimer's or Parkinson's). Modulation of a biological and/or pharmacological activity of livin, as described herein, by means of small molecule therapeutic compounds or administration of a dominant negative mutant form, or antisense molecule, or therapeutic antibody is expected to ameliorate pathophysiological conditions associated with apoptosis.